Method of cancer treatment using sirna silencing
a cancer treatment and silencing technology, applied in the field of cancer treatment using sirna silencing, can solve the problems of renewed tumor rejection, currently used immunomodulatory drugs pose potential toxic threats to humans, and no studies have been demonstrated to support such contentions, so as to reduce the expression of said ido, and reduce the effect of ido-directed tumor immunosuppression
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Animal and Cell Lines
[0098]Male C57BL / 6 and BALB / c mice were purchased from Jackson laboratories (Bar Harbour, Me.). A murine melanoma cell line established from a C57BL / 6 mouse designated B16F10 was obtained from American Type Culture Collection (ATCC, Manassas, Va.). B16 cells were cultured in RPMI1640 medium (sigma) with 10% fetal bovine serum, L-glutamine, penicillin and streptomycin at 37° C. in 5% CO2 incubator.
IDO-Specifics siRNA Expression Vectors
[0099]siRNA expression vectors were constructed using the pQuiet plasmid (Welgen Inc.). Specific IDO-siRNA inserts were designed according to manufacturer's instruction. The oligonucleotide contained a 19-mer hairpin sequences specific to the IDO mRNA target, a loop sequence separating the two complementary domains, a two nucleotide overhang at the 3′ end, a poly thymidine stretch to terminate transcription and a 5′ single-stranded overhang for ligation into the pQuiet vector using the Bgl I and Xob I cut sites....
example 2
IDO is Efficiently Silenced by siRNA in B16 Melanoma Cells
[0115]IDO has been demonstrated to be expressed in B16 melanomas. To test the efficacy of gene silencing in the B16 melanoma cell line, a liposome transfection method was incorporated to deliver our IDO-siRNA-containing pQuiet plasmid into B16 cells in vitro. Twenty four hours after transfection, potent gene silence was observed at the transcriptional level as detected by RT-PCR (FIG. 1A). The protein level of IDO was also significantly decreased as detected by Western Blotting (FIG. 1B), suggesting the expression of IDO in B16 cells could be effectively inhibited by IDO-siRNA.
[0116]Since IDO functions as a specific enzyme to primarily catabolize tryptophan, in order to characterize the effects of silencing on enzyme efficacy the change in levels of tryptophan in B16 culture medium upon silencing was detected. B16 cells silenced by siRNA displayed significantly lower IDO functionality (p<0.05) than B16 cells transfected with ...
example 3
Silencing IDO in B16 Cells Prior to Inoculation Inhibits Tumor Growth
[0117]It has been reported that IDO expression is correlated with tumor progression. Tumor cells expressing IDO produce larger and more aggressive tumors than those which have been chemically silenced of IDO expression (4). It was therefore postulated that siRNA-induced silencing of IDO in B16 cells prior to inoculation would restrain tumor growth substantially. To test this hypothesis, IDO-siRNA into B16 cells were transfected using a liposome transfection method in vitro. IDO-silenced B16 cells were then subcutaneously injected in to syngeneic C57 / BL6 mice. As demonstrated in FIG. 2A, tumor onset time in IDO-siRNA-treated mice was substantially postponed (p<0.0001) in comparison with mice injected with non-silenced or nonsense-siRNA-treated B16 cells.
[0118]In addition to tumor onset time, also observed were the sizes of tumors derived from IDO-silenced and control B16 cells. Wild-type B16 cells, as well as mock-s...
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